Oxidation and biosynthesis of fatty acids Stages of

Oxidation and biosynthesis of fatty acids

Stages of fatty acid oxidation (1) Activation of fatty acids takes place on the outer mitochondrial membrane (2) Transport into the mitochondria (3) Degradation to two-carbon fragments (as acetyl Co. A) in the mitochondrial matrix (b-oxidation pathway)

(1) Activation of Fatty Acids • Fatty acids are converted to Co. A thioesters by acyl -Co. A synthetase (ATP dependent) • The PPi released is hydrolyzed by a pyrophosphatase to 2 Pi • Two phosphoanhydride bonds (two ATP equivalents) are consumed to activate one fatty acid to a thioester

(2) Transport of Fatty Acyl Co. A into Mitochondria • The carnitine shuttle system. • Fatty acyl Co. A is first converted to acylcarnitine (enzyme carnitine acyltransferase I (bound to the outer mitochondrial membrane). • Acylcarnitine enters the mitochondria by a translocase. • The acyl group is transferred back to Co. A (enzyme carnitine acyltransferase II).

• Carnitine shuttle system • Path of acyl group in red

(3) The Reactions of b oxidation • The b-oxidation pathway (b-carbon atom (C 3) is oxidized) degrades fatty acids two carbons at a time

1. Oxidation of acyl Co. A by an acyl Co. A dehydrogenase to give an enoyl Co. A Coenzyme - FAD

2. Hydration of the double bond between C-2 and C-3 by enoyl Co. A hydratase with the 3 -hydroxyacyl Co. A (b-hydroxyacyl Co. A) formation

3. Oxidation of 3 -hydroxyacyl Co. A to 3 -ketoacyl Co. A by 3 -hydroxyacyl Co. A dehydrogenase Coenzyme – NAD+

4. Cleavage of 3 -ketoacyl Co. A by the thiol group of a second molecule of Co. A with the formation of acetyl Co. A and an acyl Co. A shortened by two carbon atoms. Enzyme -ketothiolase.

The shortened acyl Co. A then undergoes another cycle of oxidation The number of cycles: n/2 -1, where n – the number of carbon atoms

b-Oxidation of Fatty acyl Co. A saturated fatty acids

• One round of oxidation: 4 enzyme steps produce acetyl Co. A from fatty acyl Co. A • Each round generates one molecule each of: FADH 2 NADH Acetyl Co. A Fatty acyl Co. A (2 carbons shorter each round) Fates of the products of -oxidation: - NADH and FADH 2 - are used in ETC - acetyl Co. A - enters the citric acid cycle - acyl Co. A – undergoes the next cycle of oxidation

ATP Generation from Fatty Acid Oxidation Net yield of ATP per one oxidized palmitate Palmitate (C 15 H 31 COOH) - 7 cycles – n/2 -1 • The balanced equation for oxidizing one palmitoyl Co. A by seven cycles of b oxidation Palmitoyl Co. A + 7 HS-Co. A + 7 FAD+ + 7 NAD+ + 7 H 2 O 8 Acetyl Co. A + 7 FADH 2 + 7 NADH + 7 H+ ATP generated 8 acetyl Co. A 7 FADH 2 7 NADH 10 x 8=80 7 x 1. 5=10. 5 7 x 2. 5=17. 5 108 ATP expended to activate palmitate Net yield: -2 106 ATP

LIPID METABOLISM: FATTY ACID OXIDATION

b-OXIDATION OF ODD-CHAIN FATTY ACIDS • Odd-chain fatty acids occur in bacteria and microorganisms • Final cleavage product is propionyl Co. A rather than acetyl Co. A • Three enzymes convert propionyl Co. A to succinyl Co. A (citric acid cycle intermediate)

Propionyl Co. A Is Converted into Succinyl Co. A 1. Propionyl Co. A is carboxylated to yield the D isomer of methylmalonyl Co. A. The hydrolysis of an ATP is required. Enzyme: propionyl Co. A carboxylase Coenzyme: biotin

2. The D isomer of methylmalonyl Co. A is racemized to the L isomer Enzyme: methylmalonyl-Co. A racemase

3. L isomer of methylmalonyl Co. A is converted into succinyl Co. A by an intramolecular rearrangement Enzyme: methylmalonyl Co. A mutase Coenzyme: vitamin B 12 (cobalamin)

OXIDATION OF FATTY ACIDS IN PEROXISOMES Peroxisomes - organelles containing enzyme catalase, which catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen Acyl Co. A dehydrogenase transfers electrons to O 2 to yield H 2 O 2 instead of capturing the high-energy electrons by ETC, as occurs in mitochondrial oxidation.

METABOLISM OF LIPIDS: SYNTHESIS OF FATTY ACIDS

Fatty Acid Synthesis • Occurs mainly in liver and adipocytes, in mammary glands during lactation • Occurs in cytoplasm • FA synthesis and degradation occur by two completely separate pathways • When glucose is plentiful, large amounts of acetyl Co. A are produced by glycolysis and can be used for fatty acid synthesis

Three stages of fatty acid synthesis: A. Transport of acetyl Co. A into cytosol B. Carboxylation of acetyl Co. A C. Assembly of fatty acid chain

A. Transport of Acetyl Co. A to the Cytosol • Acetyl Co. A from catabolism of carbohydrates and amino acids is exported from mitochondria via the citrate transport system • Cytosolic NADH also converted to NADPH • Two molecules of ATP are expended for each round of this cyclic pathway

Citrate transport system

Sources of NADPH for Fatty Acid Synthesis 1. One molecule of NADPH is generated for each molecule of acetyl Co. A that is transferred from mitochondria to the cytosol (malic enzyme). 2. NADPH molecules come from the pentose phosphate pathway.

B. Carboxylation of Acetyl Co. A Enzyme: acetyl Co. A carboxylase Prosthetic group - biotin A carboxybiotin intermediate is formed. ATP is hydrolyzed. The CO 2 group in carboxybiotin is transferred to acetyl Co. A to form malonyl Co. A. Acetyl Co. A carboxylase is the regulatory enzyme.

C. The Reactions of Fatty Acid Synthesis • Five separate stages: (1) Loading of precursors via thioester derivatives (2) Condensation of the precursors (3) Reduction (4) Dehydration (5) Reduction

During the fatty acid synthesis all intermediates are linked to the protein called acyl carrier protein (ACP-SH), which is the component of fatty acyl synthase complex. The pantothenic acid is a component of ACP. Intermediates in the biosynthetic pathway are attached to the sulfhydryl terminus of phosphopantotheine group.

The elongation phase of fatty acid synthesis starts with the formation of acetyl ACP and malonyl ACP. Acetyl transacylase and malonyl transacylase catalyze these reactions. Acetyl Co. A + ACP acetyl ACP + Co. A Malonyl Co. A + ACP malonyl ACP + Co. A

Condensation reaction. Acetyl ACP and malonyl ACP react to form acetoacetyl ACP. Enzyme acyl-malonyl ACP condensing enzyme.

Reduction. Acetoacetyl ACP is reduced to D-3 hydroxybutyryl ACP. NADPH is the reducing agent Enzyme: -ketoacyl ACP reductase

Dehydration. D-3 -hydroxybutyryl ACP is dehydrated to form crotonyl ACP (trans- 2 -enoyl ACP). Enzyme: 3 -hydroxyacyl ACP dehydratase

Reduction. The final step in the cycle reduces crotonyl ACP to butyryl ACP. NADPH is reductant. Enzyme - enoyl ACP reductase. This is the end of first elongation cycle (first round).

In the second round butyryl ACP condenses with malonyl ACP to form a C 6 - -ketoacyl ACP. Reduction, dehydration, and a second reduction convert the C 6 - ketoacyl ACP into a C 6 acyl ACP, which is ready for a third round of elongation.

Final reaction of FA synthesis • Rounds of synthesis continue until a C 16 palmitoyl group is formed • Palmitoyl-ACP is hydrolyzed by a thioesterase Overall reaction of palmitate synthesis from acetyl Co. A and malonyl Co. A Acetyl Co. A + 7 Malonyl Co. A + 14 NADPH + 14 H+ Palmitate + 7 CO 2 + 14 NADP+ + 8 HS-Co. A + 6 H 2 O

Organization of Multifunctional Enzyme Complex in Eukaryotes The synthase is dimer with antiparallel subunits. Each subunit has three domains. ACP is located in domain 2. Domain 1 contains transacylases, ketoacyl-ACP synthase (condensing enzyme) Domain 2 contains acyl carrier protein, -ketoacyl reductase, dehydratase, and enoyl reductase. Domain 3 contains thioesterase activity.

Fatty Acid Elongation and Desaturation The common product of fatty acid synthesis is palmitate (16: 0). Cells contain longer fatty acids and unsaturated fatty acids they are synthesized in the endoplasmic reticulum. The reactions of elongation are similar to the ones seen with fatty acid synthase (new carbons are added in the form of malonyl Co. A). For the formation of unsaturated fatty acids there are various desaturases catalizing the formation of double bonds.

THE CONTROL OF FATTY ACID METABOLISM Acetyl Co. A carboxylase plays an essential role in regulating fatty acid synthesis and degradation. The carboxylase is controlled by hormones: Ø glucagon, Ø epinephrine, and Ø insulin. Another regulatory factors: Ø citrate, Ø palmitoyl Co. A, and Ø AMP

Global Regulation is carried out by means of reversible phosphorylation Acetyl Co. A carboxylase is switched off by phosphorylation and activated by dephosphorylation Insulin stimulates fatty acid synthesis causing dephosphorylation of carboxylase. Glucagon and epinephrine have the reverse effect (keep the carboxylase in the inactive phosphorylated state). Protein kinase is activated by AMP and inhibited by ATP. Carboxylase is inactivated when the energy charge is low.

Local Regulation Acetyl Co. A carboxylase is allosterically stimulated by citrate. The level of citrate is high when both acetyl Co. A and ATP are abundant (isocitrate dehydrogenase is inhibited by ATP). Palmitoyl Co. A inhibits carboxylase.

Fed state: Response to Diet • Insulin level is increased • Inhibits hydrolysis of stored TGs • Stimulates formation of malonyl Co. A, which inhibits carnitine acyltransferase I • FA remain in cytosol (FA oxidation enzymes are in the mitochondria) Starvation: • Epinephrine and glucagon are produced and stimulate adipose cell lipase and the level of free fatty acids rises • Inactivate carboxylase, so decrease formation of malonyl Co. A (lead to increased transport of FA into mitochondria and activate the b-oxidation pathway)